Venous thrombosis and pulmonary embolism are major causes of morbidity and mortality in the United States, accounting for about 270,000 hospitalizations a year (Anderson, F. A., Jr. et al., Arch. Intern. Med. 151:933-938 (1991)). In addition, it is estimated that about 50,000-200,000 patients a year die from pulmonary embolism (Lilienfeld, D. E. et al., Chest 98:1067-1072 (1990)). In surprising contrast with the mortality rate for myocardial infarction, the mortality rate for pulmonary embolism (estimated at 9.2% in treated patients) has not improved in the last 30 years (Lilienfeld, D. E. et al., Chest 98:1067-1072 (1990); Giuntini, C. et al., Chest 107:3S-9S (1995)). Moreover, survivors of venous thromboembolism are known to be at risk for recurrent thrombosis, postphlebitic syndrome, and pulmonary hypertension (Sutton, G. C. et al., Br. Heart J. 39:1135-1192 (1977); Salzman, E. W. and Hirsch J., "The Epidemiology, Pathogenesis and Natural History of Venous Thrombosis," in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, Coleman, R. W. et al., eds., 3rd ed. Philadelphia, Pa. (1994), pp. 1275-1296).
A. Mechanism of Clot Formation and Lysis PA0 B. Treatment for Venous Thrombosis and Pulmonary Embolism PA0 C. Alpha-2 antiplasmin Antibodies
Clots (or thrombi in a patient) are composed of fibrin and blood platelets in various ratios. The fundamental reaction in blood clotting involves the conversion of a soluble plasma protein (fibrinogen) into insoluble fibrin. The conversion of fibrinogen into fibrin is catalyzed by the enzyme, thrombin, which is a serine protease.
Clot lysis is mediated by plasmin. Under natural conditions, plasminogen is converted to plasmin by plasminogen activators. Natural plasmin inhibitors include .alpha.2-antiplasmin, .alpha.2-macroglobulin and .alpha.-1-antitrypsin, all glycoproteins. Alpha-2-antiplasmin has a much higher affinity for plasmin than .alpha.2-macroglobulin and binds specifically to plasmin in a 1:1 ratio. The larger pool of .alpha.-macroglobulin acts as a reservoir inhibitor (Kane K. K., Ann. Clin. Lab. Sci. 14:443-449 (1984)). Thus, clot lysis by the administration of t-PA is limited by the rapid and irreversible inactivation of plasmin by plasmin inhibitors.
Standard therapy for venous thromboembolism is heparin, which potentiates thrombin and factor Xa inhibition by antithrombin III (Goldhaber, S., Chest 107:45S-51S (1995)). Although heparin decreases new thrombus (clot) S formation, clinical studies suggest that there is little early endogenous lysis of the large thrombi that often exist at the time of diagnosis in patients with venous thromboembolism (Goldhaber, S. Z. et al., Lancet 2:886-889 (1986); "The Urokinase Pulmonary Embolism Trial," Circulation 47:1-108 (1973); Goldhaber, S. Z. et al., Am. J. Med. 88:235-240 (1990); Goldhaber, S. Z. et al., Lancet 341:507-511 (1993)). Since large thrombi are associated with an increase in morbidity and mortality, several studies have examined the effects of plasminogen activators in patients with venous thromboembolism (Goldhaber, S. Z. et al., Lancet 2:886-889 (1986); "The Urokinase Pulmonary Embolism Trial," Circulation 47:1-108 (1973); Goldhaber, S. Z. et al., Am. J Med 88:235-240 (1990); Goldhaber, S. Z. et al., Lancet 341:507-511(1993)).
Compared with heparin alone, plasminogen activators cause significant increases in the lysis of venous thromboemboli, but patients are frequently left with large amounts of residual thrombi in the lungs or deep veins immediately after therapy (Goldhaber, S. Z. et al., Lancet 2:886-889 (1986); "The Urokinase Pulmonary Embolism Trial," Circulation 47:1-108 (1973); Goldhaber, S. Z. et al., Am. J. Med. 88:235-240 (1990); Goldhaber, S. Z. et al., Lancet 341:507-511 (1993)). None of the randomized, controlled trials of patients with pulmonary embolism have demonstrated a mortality benefit from plasminogen activators, although this may well be due to the small numbers of patients enrolled in these studies. Use of plasminogen activators for myocardial infarctions has shown that 45-70% of patients with coronary thrombosis have failed to achieve full 90 minutes reperfusion with these agents.
Why venous thromboemboli resist fibrinolysis is unknown. Physical characteristics such as size, retraction, exposure to blood flow, and age may affect the lysis of these large fibrin-rich thrombi (Prewitt, R. M., Chest 99:157S-164S (1991)). However, it is also likely that the fibrinolytic resistance of these thrombi is regulated by specific molecular factors such as factor XIII, plasminogen activator inhibitor 1 (PAI-1), and alpha-2-antiplasmin (.alpha.2AP) (Collen, D., Eur. J. Biochem. 69:209-216 (1976); Moroi, M. and Aoki, N., J. Biol. Chem. 251:5956-5965 (1976); Mullertz, S. and Clemmensen, I., Biochem. J. 159:545-553 (1976); Sakata, Y. and Aoki, N., J. Clin. Invest. 69:536-542 (1982); Robbie, L. A. et al., Thromb. Haemostas. 70:301-306 (1993); Francis, C. W. and Marder, V. J., J. Clin. Invest. 80:1459-1465 (1987); Jansen, J. W. C. M. et al., Thromb. Haemostas. 57:171-175 (1987); Reed, G. L. et al., Trans. Assoc. Am. Phys. 104:21-28 (1991); Stringer, H. A. and Pannekoek, H., J. Biol. Chem. 270:11205-11208 (1995); Carmeliet, P. et al., J. Clin. Invest. 92:2756-2760 (1993); Lang, I. M. et al., Circulation 89:2715-2721 (1994); Marsh, J. J. et al., Circulation 90:3091-3097 (1994)).
Because .alpha.2AP is an ultrafast covalent inhibitor of plasmin (the enzyme that degrades thrombi), .alpha.2AP is a particularly likely cause of thrombus resistance (Collen, D., Eur. J. Biochem. 69:209-216 (1976); Moroi, M. and Aoki, N., J. Biol. Chem. 251:5956-5965 (1976); Mullertz, S. and Clemmensen, I., Biochem. J. 159:545-553 (1976)). Moreover, .alpha.2AP is the only fibrinolytic inhibitor that is covalently crosslinked to the fibrin surface (Sakata, Y. and Aoki, N., J. Clin. Invest. 69:536-542 (1982)). This crosslinking (by activated factor XIII) concentrates .alpha.2AP on the fibrin surface, where it inhibits the initiation of fibrinolysis (Sakata, Y. and Aoki, N., J. Clin. Invest. 69:536-542 (1982)). Previous in vitro studies have shown that clots from .alpha.2AP-deficient patients lyse spontaneously, suggesting that .alpha.2AP plays a critical role in thrombus resistance to endogenous plasminogen activators (Aoki, N. et al., Blood 62:1118-1122 (1983); Miles, L. A. et al., Blood 59:1246-1251 (1982)). These observations led to the hypothesis that .alpha.2AP is a molecular mediator of the thrombus resistance seen in patients with pulmonary embolism. To test this hypothesis, we generated a specific inhibitor of .alpha.2AP and used it to determine the role played by .alpha.2AP in the regulation of lysis of experimental pulmonary emboli.
If an individual has formed a fibrin clot (thrombus) prior to the availability of medical assistance, the clot may be dissolved through the use of agents capable of lysing the fibrin thrombus, and thereby permitting blood to again flow through the affected blood vessel. Such agents include plasmin, anti-coagulants (such as, for example, heparin, hirudin and activated protein C), plasminogen activators (such as, for example, streptokinase, prourokinase, urokinase, tissue-type plasminogen activator, staphylokinase, and vampire bat plasminogen activator), and other such agents (Ganz, W. et al., J. Amer. Coll. Cardiol. 1:1247-1253 (1983); Rentrop, K. P. et al., Amer. J. Cardiol. 54:29E-31E (1984); Gold, H. K. et al., Amer. J. Cardiol. 53:122C-125C (1984)).
At present, treatment of pulmonary embolism, myocardial infarction, thrombosis, and stroke is partially achieved through the administration of thrombolytic agents. Use of such agents in therapy often results in incomplete lysis, and promotes the reformation of thrombi and reocclusion of the affected blood vessels. Hence, a need exists for an improvement in thrombolytic therapy which enhances fibrinolysis, while minimizing fibrinogen breakdown and preventing reformation of thrombi.
Alpha-2-antiplasmin (.alpha.2AP) has three functional domains: the reactive site for plasmin, the plasmin(ogen) or LBS-binding site [complementary to the LBS (lysine-binding site) of plasmin(ogen)], and the crosslinking site for fibrin. Mimuro, J. et al., Blood 69:446-453 (1987). Mimuro et al. discloses antibodies to .alpha.2AP, one of which (JPTI-1) was specific to the reactive site of .alpha.2AP and prevented formation of .alpha.2AP complexes, thereby inhibiting antiplasmin activity. However, Mimuro et al. does not teach administration of the JPTI-1 antibody to enhance clot lysis. Other antibodies specific for .alpha.2AP are taught by Plow, E. F. et al., J. Biol. Chem. 255:2902-2906 (1980); Wimen, B. et al., Scan. J. Clin. Lab. Invest. 43:27-33 (1983); Hattey, E. et al., Thromb. Res. 45:485-495 (1987); Collen, U.S. Pat. No. 4,346,029 (1980); and Collen, U.S. Pat. No. 4,198,335 (1980).